胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术
Summary
该协议使读者能够分析胆汁盐诱导的肠道致病菌的生物膜形成, 采用多层面的方法来通过评估附着体、胞外高分子物质基质形成来捕获细菌生物膜的动态特性,和分散。
Abstract
生物膜形成是一种动态的、多级的过程, 在恶劣的环境条件下或受压力的时代发生在细菌中。对于肠道病原体, 在胃肠道转运和胆汁暴露过程中, 引起了明显的应力反应, 这是人类消化的正常成分。为了克服胆汁的杀菌作用, 许多肠道病原体形成生物膜假说, 允许在小肠中转时存活。在这里, 我们提出了定义生物膜形成的方法, 通过固相粘附测定, 以及胞外高分子物质 (EPS) 矩阵检测和可视化。此外, 还提出了生物膜色散评估法, 模拟了在感染过程中引发细菌释放的事件的分析。在高通量的96井板粘附试验中, 用结晶紫染色法检测粘附细菌。eps 生产评价由两种方法确定, 即 eps 基质的显微染色和荧光共轭多糖结合凝集素的半定量分析。最后通过菌落计数和电镀来测量生物膜的分散性。来自多种化验的阳性数据支持生物膜的特性, 可用于鉴别其他细菌菌株中的胆汁盐诱导生物膜的形成。
Introduction
生物膜形成是恶劣环境条件下引起的一种重要的细菌生存策略。暴露在杀菌化合物如抗生素或营养或氧的变化, 导致细菌的压力状态, 可以通过生物膜形成缓解。生物膜的特点是细菌附着于表面或其他细菌, 并伴随着一个 EPS 矩阵的分泌, 主要由多糖1,2,3。生物膜形成是一个动态过程, 其中一连串事件的高潮形成一个成熟的黏附细菌社区1,2,3。细菌产生 adhesins, 以方便早期附着, 而转移黏附基因表达谱, 以加强附着在生物膜成熟。同时, EPS 生产发生在基质中覆盖细菌群落, 以保护细胞不受初始压力源的侵害。生物膜内含有的细菌生长缓慢;因此, 大多数抗生素都是无效的。此外, 缓慢的增长节省能源, 直到条件改变有利于细菌生长1,2,3。在严酷的条件已经过去了, 细菌分散生物膜和恢复浮游生活方式1,2,3。传统上, 生物膜被观察在表面上, 并代表了持续的临床挑战, 因为感染水库存在于导管和居住设备1,2,3。
最近对几种肠道病原体的生物膜形成进行了描述;感染小肠或结肠的细菌4。对于志贺菌物种,感染发生在人类结肠经胃肠道的大部分转运后。在通过小肠的通道中,志贺菌暴露于胆汁中;一种脂质降解的洗涤剂分泌到肠道, 以促进脂肪的消化, 同时杀死大多数细菌5。肠道病原体具有抵御胆汁6杀菌效果的独特能力。我们最近的分析利用了体内类似的葡萄糖和胆汁盐的组合, 以显示在S. 福氏以及其他种类的志贺菌,致病性大肠杆菌, 和 沙门氏菌4。此前,沙门氏菌 enterica钩伤寒被证明形成胆汁诱导生物膜, 由于在慢性感染期间胆囊的独特殖民化7,8,9, 10. 此外, 以前的研究与弧菌11和弯曲的12表明生物膜形成的反应胆汁。因此, 分析将胆汁诱导的生物膜形成观察扩展到其他病原体, 有助于建立一种保守的肠道病原体对胆汁的反应。不同于慢性生物膜, 其中细菌基因转录是有限的, 细胞衰老可能发生1,2,3, 我们建议, 肠道胆汁诱导生物膜是更短暂的性质。这种瞬态, 剧毒生物膜是虚幻快速拆卸 (如在弥散检测中看到) 和增强的毒力基因表达观察在生物膜的人口4,6。
由于生物膜的形成是一个多方面的, 动态的过程和使用胆汁盐作为一个起始因素最近才被描述的大多数肠道病原体, 使用的工具和技术是独特的和创造性的应用传统方法。因此, 这里提出了三免费战略, 以量化的几个重要特征的胆汁盐诱导生物膜形成, 包括细菌黏附, 生产的 EPS 基质, 和分散的活菌从生物膜。这些技术主要用于研究与志贺菌;因此, 评估其他肠道病原体可能需要优化。然而, 来自所有三种化验的阳性数据都支持生物膜的鉴定, 并建立了胆汁盐诱导生物膜形成的可再生的协议。
Protocol
1. 试剂的制备 胆汁盐培养基: 制备含有0.4% 胆汁盐 (重量/容积) 的胰蛋白酶大豆汤 (并用重悬), 50 毫升蒸压的200毫克胆汁盐。使用0.22 µm 过滤器进行杀菌消毒。每周制作新鲜媒体。注: 经常使用的胆汁盐是1:1 混合胆酸钠和脱氧钠, 从绵羊和牛胆囊分离。如前所示的4, 胆汁盐诱导的生物膜形成需要葡萄糖的存在。Luria-Bertani (磅) 汤增加了葡萄糖;因此, 在志贺菌和其他?…
Representative Results
在图 1中, 在含有胆汁盐的培养基的生长过程中, 大多数六种肠道病原体均诱导生物膜形成。在几乎所有的菌株中都能观察到胆汁盐暴露后黏附细菌的显著增加。异常是 enteroaggregative大肠杆菌(EAEC);但是, 请注意对Δ aaf突变4的诱导观察。结果表明, 在没有聚集黏附放线菌菌毛 I (AAF)21 的情况下, EAEC …
Discussion
由于生物膜的动态性质以及菌株、材料、实验室和化验结果之间的变异性, 生物膜形成的分析具有挑战性。本文提出了在胆盐暴露后肠道病原体中生物膜形成的几种策略, 并对其进行了实验研究, 以促进重现性。还有其他的考虑, 以确保重现性。首先, 我们建议执行至少三个独立的实验, 每个与技术 triplicates, 以确认观察和统计意义, 由于可能发生的变化。第二, 使用积极和消极的控制是至关重要的, ?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢雷切尔 b. Chanin 和利亚诺斯的技术援助。我们感谢安东尼 t. Maurelli, 布莱恩 p. 赫尔利, 塞科法萨诺, 布雷特 e Swierczewski, 和鲍比 Cherayil 为本研究中使用的菌株。这项工作得到了国家过敏和传染性疾病研究所赠款 K22AI104755 (脑脊液) 的支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。
Materials
| Tryptic Soy Broth | Sigma-Aldrich | 22092-500G | |
| Crystal Violet | Sigma | C6158-50 | |
| Concanavalin-A FITC | Sigma | C7642-10mg | |
| Glucose | Sigma | G7021-1KG | |
| Bile Salts | Sigma | B8756-100G | |
| LB Agar | Sigma | L7533-1KG | |
| 14 mL culture tubes, 17 x 100 mm, plastic, sterile | Fisher | 14-959-11B | |
| Vectashield hard-set antifade with DAPI | Vector Laboratories | H-1500 | |
| Formaldehyde | Sigma-Aldrich | F1635-500 | |
| Gluteraldehyde | Sigma-Aldrich | G6257 | |
| Flat-bottomed 96-well plates (clear) | TPP | 92696 | |
| Flat-bottomed 96-well plates (black) | Greiner Bio-One | 655076 | |
| Flat-bottomed 24-well plates (clear) | TPP | 92424 | |
| Glass coverslips 12mm, round | Fisher | 08-774-383 | |
| 96-well plate reader | Spectramax | ||
| Flourescent plate reader | Biotek Synergy 2 | ||
| Confocal or Fluorescent Microscope | Nikon A1 confocal microscope | ||
| 37°C Shaking Incubator | New Brunswick Scientific Excella E25 | ||
| 37°C Plate Incubator | Thermolyne Series 5000 |
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